Chemosphere 118 (2015) 253–260
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Changes of polybrominated diphenyl ether concentrations in ducks with background exposure level and time Peng-Yan Liu a, Xiao-Ran Chen a,b, Ya-Xian Zhao b, Yuan-Yuan Li b, Xiao-Fei Qin b, Zhan-Fen Qin b,⇑ a b
College of Chemistry and Environmental Science, Hebei University, Baoding, Hebei, China State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
h i g h l i g h t s We conducted a semi-field experiment of ducks in an e-waste recycling site.
P PBDEs increased in fat with time, but fluctuated in other tissues with exposure level. High brominated DE levels increased with time but low brominated DE fluctuated in fat. High brominated DEs seem to have longer half-lives than low brominated DEs in fat.
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 2 September 2014 Accepted 11 September 2014 Available online 4 October 2014 Handling Editor: H. Fiedler Keywords: Polybrominated diphenyl ethers Accumulation Ducks Time Exposure levels
a b s t r a c t To reveal what degree bioaccumulation of polybrominated diphenyl ethers (PBDEs) depends on exposure time and other factors, we conducted a semi-field experiment for a year (June 2008–June 2009) in a village in an e-waste recycling site in Taizhou, China. Approximately one hundred of juvenile ducks (Anas domestica Linnaeus) were entrusted to a villager. The ducks lived and forged in a PBDE-polluted pond from the late March to the end of November. Fish and mudsnails that were heavily polluted by PBDEs were main food. In cold days (from December to the middle March), the ducks lived in the villager’ house, and mainly fed on paddy, which contained lower concentrations of PBDEs than fish and mudsnails. The P female ducks were sampled for PBDE analysis every three months. We found that the PBDE concentrations in duck liver, muscle, lung and brain fluctuated greatly with the changes of exposure levels that P were determined by the environment and diets, but the PBDE concentrations in fat tissue increased P PBDE successively with time. Congener analysis demonstrated that the successive increase in the concentrations with time in fat tissue was due to the successive increase in BDE-209, -183 and -153 concentrations, with large fluctuations of low brominated congeners. The results show that PBDE concentrations in liver, muscle, lung and brain tissues heavily depends on exposure levels rather than exposure time. In fat tissue, by contrast, PBDE concentrations (mainly high brominated congeners) slightly depends on exposure levels but heavily depend on time relative to other tissues, implying that high brominated congeners seem to have longer half-lives than low brominated congeners in fat tissue. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Polybrominated diphenyl ethers (PBDEs) are a class of widely used flame retardants in various products, including in textiles, polyurethane foams, thermoplastics and electronic products. Due to high levels of production and application, PBDEs have become ubiquitous environmental pollutants in the environment (Rahman et al., 2001; De Wit, 2002). PBDEs are also found in animals and human tissues because of their hydrophobicity and bioaccumula⇑ Corresponding author. Tel.: +86 10 6291 9177; fax: +86 10 6292 3563. E-mail address: [email protected] (Z.-F. Qin). http://dx.doi.org/10.1016/j.chemosphere.2014.09.037 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
tion (McDonald, 2002; Darnerud, 2008). Animal studies have shown that PBDEs have a wide spectrum of toxicological effects, including thyroid disruption, developmental neurotoxicity, reproduction, immunotoxicity, etc. (Damerud, 2003). There are increasing epidemiological evidences that PBDEs might be associated with some parameters on human health (Chen et al., 2011; Chevrier et al., 2011; Bradman et al., 2012; Eskenazi et al., 2013). Due to bioaccumulation and potential toxicity, thus, PBDEs have aroused great concern. Deca-BDE products have still been produced and used widely, although commercial tetra- and octa-BDEs were added to Annex A of the Stockholm Convention PBDEs in 2009 (Alaee et al., 2003; Covaci et al., 2011).
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P.-Y. Liu et al. / Chemosphere 118 (2015) 253–260
Numerous studies have demonstrated that the concentrations of some persistent organic pollutants (POPs), such as polychlorinated biphenyl (PCB), DDT, DDE, increase generally with exposure time or age in biotic tissues (Ronald et al., 1984; Johnson-Restrepo et al., 2005; Munoz-De-Toro et al., 2006; Mcgraw and Waller, 2009; Weijs et al., 2009; Goutner et al., 2011). However, a few of studies have demonstrated that PBDE bioaccumulation seems to increase with age in some biotic tissues (Riget et al., 2006; Kim et al., 2012), whereas some biomonitoring data show no association between PBDEs concentrations and time/age in biotic samples, such as fish (Zhang et al., 2010; Vigano et al., 2011), mammals (Rayne et al., 2004; Muir et al., 2006; Weijs et al., 2009), and humans (Covaci et al., 2008; Harrad and Porter, 2007; Kunisue et al., 2007). Overall, what degree PBDE bioaccumulation depends on exposure time and other factors has been unclear. Taizhou in Zhejiang Province is one of the largest electric waste (e-waste) recycling areas in China. Recycling of e-waste using primitive methods has resulted in heavy PBDE pollution in the environment, although the primitive e-waste recycling has been controlled. In our previous studies, high concentrations of PBDEs were found in soils, sediments, animal tissues and human samples from some e-waste recycling sites in Taizhou (Liang et al., 2008; Yang et al., 2008; Zhao et al., 2009; Liu et al., 2011). To study what degree PBDE bioaccumulation depends on exposure time and other factors, in this study, we conducted a semi-field experiment in an e-waste recycling site in Taizhou to investigate changes in PBDE concentrations in ducks within a year and discuss crucial factors for PBDE accumulation. 2. Materials and methods 2.1. Chemicals BDE-71 and 13C-labled BDE-209 as surrogate standards, and a standard solution of PBDE congeners (EO-5278, BDE-28, -47, -99, -100, -153, -154, -183, -209.) were obtained from Cambridge Isotope Laboratories (USA). Methylene dichloride, n-Hexane, and nonane were of pesticide grade and purchased from Tedia (USA). 2.2. Semi-field experiment on ducks During June 2008–June 2009, we conducted a semi-field experiment in a village in Taizhou, where a mass of e-wastes from abroad as well as home were disassembled and shattered into powder to obtain the usable materials, and discarded e-waste powder was stacked around this village. We purchased about one hundred of one-month-old ducks (Anas domestica Linnaeus) from a farm (no factory and e-waste recycling site in the surrounding) in Zhejiang, and entrusted a villager in this village with them. The villager’ house was approximately 500 m away from the foot of a hill, where a mass of e-waste powder was stacked. There was a pond in the front of the villager’ house. When raining, rain water from the foot of the hill flew into the pond. From the late March to the end of November, the ducks forged in the pond at daytime, and went back to their nest in the villager’ house at night. In the pond, there were a lot of mudsnails and fish, which were good food for the ducks. In cold days, from December to the early March, these ducks lived in the villager’ house, and they mainly fed on paddy. The semi-field experiment began on June 15, when we sampled 3 ducks to examine background PBDE levels in the ducks. On September 15 (the 3rd month), December 15 (the 6th month), March 15 (the 9th month) and the next June 15 (the 12th month), the ducks were sampled for PBDE analysis. Because most ducks died in the first month after the beginning of the experiment, we only sampled 3 female ducks each time. The ducks sampled in the 3rd month never laid eggs, whereas the ducks sampled in the
6th, 9th and 12th month laid eggs. As live ducks were transferred to the laboratory, they were anesthetized and weighed. Liver, lung, muscle, fat and brain tissues were collected. All samples were cleaned with deionized water, weighed and wrapped in aluminum foil twice, and sealed in plastic bags to minimize the possibility of contamination. Then, samples were stored at 20 °C in darkness until analysis. At the beginning of the semi-field experiment, we collected the water, sediment, mudsnail and fish samples from this pond. The water samples (40 L) were filtered through glass fiber filters (0.8 lm pore size) and the filtrate was passed through a XAD-2 resins column (Supelco, Bellfonte, USA), which was pre-extracted with methanol, dichloromethane and hexane, to enrich PBDEs on-site. Then, the XAD-2 resins column and sediment samples were stored in a cool box and transported to laboratory. The eluent was concentrated to about 2 mL in a rotary evaporator. Then the sample solution was transferred to a vial and reduced to a volume of 20 lL under a gentle stream of N2. The sediment samples were stored at 4 °C in darkness until analysis. Sediment samples were taken from approximately the top 2 cm of the sediment and each was constituted by five subsamples. The sediment samples (n = 3) were weighed and wrapped in aluminum foil and sealed in plastic bags on-site. All fish and mudsnails sampled in the pond were transferred to the laboratory for PBDE analysis. In the laboratory, the fish was rinsed with deionized water for three times, weighed and stored at –20 °C in darkness until analysis. After washed with deionized water, live mudsnails were placed in cool fresh water to expel the sediment and sand inside their body. The fresh water was replaced five times within 24 h. After the shells of mudsnails were removed, the samples were washed again, weighed and stored at 20 °C in the dark until analysis. Meanwhile, the rice as duck diet was also collected for PBDE analysis. 2.3. Extraction The tissue samples were freeze-dried and homogenized with anhydrous sodium sulfate, then spiked with BDE-71, and 13 C-labled BDE-209. The samples were subsequently extracted by ultrasonic extraction for twice with 30 mL of n-hexane/dichloromethane (1:1, vol/vol). Then the combined extracts were evaporated to dryness for gravimetric determination of extracted lipid content. The concentrated extract was cleaned by passing through a 15 mm i.d. column, which was packed, from the bottom to top, with 1 g activated silica gel, 3 g alkaline silica gel (33% sodium hydroxide, w/w), 1 g activated silica gel, 4 g acid silica gel (44% concentrated sulfuric acid, w/w), 4 g acid silica gel (22% concentrated sulfuric acid, w/w), 1 g activated silica gel, and 2 cm anhydrous sodium sulfate. The extract was eluted with 100 mL of hexane, concentrated to 2 mL using a rotary evaporator, transferred, and finally concentrated to approximately 0.1 mL under a gentle nitrogen stream. The final extract was transferred to GC vials. Throughout the extraction, cleanup and analysis procedures, the analyte was protected from light by wrapping the containers with aluminum foil or by using amber glassware. 2.4. PBDE analysis All sample extracts were analyzed by Agilent 6890 series gas chromatograph coupled with Agilent 5973 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) using negative chemical ionization (NCI) source in the selected ion monitoring (SIM) mode. The gas chromatography column was DB-5 MS fused silica capillaries (15 m, 250-lm inner diameter, 0.25 lm film thickness). The injector and interface temperature were 265 and 300 °C, respectively. The samples were injected in the pulsed splitless mode.
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P.-Y. Liu et al. / Chemosphere 118 (2015) 253–260
Methane was used as the chemical ionization moderating gas and helium as the carrier gas at a flow rate of 1.0 mL min1. The GC oven temperature program was carried out as follows: start at 100 °C, held for 1 min, increased to 200 °C at 10 °C min1, and then to 300 °C at 20 °C min1, held for 20 min. The ions of m/z 79 and 81 were monitored for BDE-28, -47, -100, -99, -154, -153, -183 and m/z 486.7 and 488.7 for BDE-209. 2.5. Quality assurance/quality control To avoid potential sample contamination, cross contamination, and PBDE degradation, proper handling was adopted from sample collection to chemical analysis. One procedural blank was run for every batch of nine samples to check the potential contamination from analysis process. Instrumental quality control was done by regular injection of solvent blanks and standard solutions. Recoveries of surrogate standards BDE-71 and 13C-labled BDE-209 were on average 84 ± 12% and 65 ± 8%, respectively. Analyte value of BDE-209 was corrected for the recovery of 13C-labled BDE-209, and the other congeners were corrected for the recovery of BDE-71. The limit of detection (LOD) was defined as the concentration of analyte in the sample producing a peak with the ratio of signal to noise of 3 (peak-to-peak). Non-detect or below LOD values were estimated as zero for the purpose of calculating totals and means. For the tissue samples, LODs ranged from 0.005 to 0.025 ng g1 wet weight (wet wt) for tri- to hepta- BDE, and 0.03 ng g1 wet weight for deca-BDE.
The average concentrations of PBDEs in fish and mudsnails in the study pond were 158.40 ng g1 wet wt and 3.40 ng g1 wet wt, respectively, while the average PBDE concentration in paddy was 4.14 ng g1 dry weight. The ducks in the semi-wild experiment would absorb much PBDEs via the uptake of fish and mudsnails and direct contact with the water and sediment in the study pond relative to via the uptake of paddy, although we could not measure daily intake of PBDEs by the ducks. There was a large difference in congener profiles of PBDEs between environmental samples and biotic samples from the study pond (Fig. 1). BDE-209 was the dominant congener in sediment and water, in particular, BDE-209 contributed 73.31% of the total PBDEs in water. However, low brominated congeners (BDE-28, -47, -99) were main congeners, whereas high brominated congeners (BDE-209 and BDE-183) were less in fish and mudsnails. The observation is consistent with previous reports that aquatic organisms accumulated much low brominated PBDE congeners and less high brominated congeners from the environment with more high brominated congeners and less low brominated congeners was consistent with previous reports (Sellstrom et al., 1998; Boon et al., 2002; Guo et al., 2008). In paddy samples as duck diets, BDE-209 accounted for approximately 80% of the total PBDEs. Thus, the ducks can absorb much high brominated PBDE congeners by direct contact with the sediment and water in the pond. On the contrary, the ducks can absorb much low brominated congeners by the intake of fish and mudsnails. In addition, the ducks can also absorb much BDE-209 by local paddy. 3.2. Growth of ducks and changes in tissues within a year
2.6. Data analysis All the statistical analysis was performed using SPSS software version 16.0 (SPSS Inc., Chicago, USA). The difference in body weight was tested by one-way analysis of variance. The concentraP tions of PBDEs and each congener in each tissue for adjacent sampling times were analyzed by t-test. P < 0.05 was regarded as statistically significant. 3. Results and discussion 3.1. PBDE levels and congener profiles in the surroundings and diets for ducks Table 1 shows the concentrations of PBDEs in water (dissolved phase and particulate phase), sediment, fish and mudsnails in the study pond as well as paddy samples as duck diets from the ewaste recycling site. The PBDE concentrations in sediment (8551.04 ng g1 dry weight) and water (42.00 ng L1) were much higher than the general levels reported in previous studies (Hale et al., 2003; Law et al., 2003; Yogui and Sericano, 2009). High values of PBDEs detected in water and sediment indicated that e-waste recycling resulted in PBDE pollution in this study area.
P PBDE concentrations in various
The ducks rapidly grew up before the first sampling. No significant difference in body weight was found among ducks collected for four sampling times, despite body weight of the ducks collected for the third time was slightly low (Table 2). The ducks sampled in the 3rd month never laid eggs, but there were some developing eggs in the abdomens. The ducks sampled in the 6th, 9th and 12th month P laid eggs. Fig. 2 and Table 2 shows the PBDE concentrations (ng g1 lipid weight) in fat, liver, lung, muscle, and brain tissues of the ducks. Statistical analysis (t-test) of the data for adjacent P sampling times revealed a rough fluctuation of PBDE concentrations with time. Before the beginning of the semi-field experiment, the ducks had low body burden of PBDEs. After 3 months, the P PBDE concentrations in ducks increased dramatically. The P PBDE concentrations in liver, muscle, lung and brain tissues flucP tuated within a year. In detail, the PBDE concentrations increased in the 6th month relative to the 3rd month, decreased in the 9th month relative to the 6th month, then, increased again during the last 3 months of the semi-field experiment. It is generally believed that the growth possibly results in decreases in pollutant concentrations in the body, so called biodilution (Hammer et al., 1993; Blais et al., 2003). Considering the lack
Table 1 PBDE concentrations (mean ± SD) in water (ng L1, n = 3), sediment (ng g1 dry weight, n = 3), fish (ng g1 wet weight, n = 10), mudsnail (ng g1 wet weight, n = 10) and paddy (ng g1 dry weight, n = 3) samples from an e-waste recycling site. Compound
Water
Sediment
Fish
Mudsnail
Paddy
BDE-28 BDE-4 BDE-99 BDE-100 BDE-153 BDE-154 BDE-183 BDE-209 P PBDEs
0.92 ± 0.08 4.89 ± 0.34 3.80 ± 0.27 0.16 ± 0.01 0.81 ± 0.04 0.28 ± 0.01 0.35 ± 0.02 30.79 ± 2.26 42.00 ± 3.42
263.90 ± 34.21 1972.30 ± 212.37 1805.90 ± 193.38 132.80 ± 15.26 562.00 ± 67.34 189.90 ± 19.47 621.20 ± 78.17 3003.00 ± 483.96 8551.04 ± 921.14
30.83 ± 2.46 100.82 ± 13.48 18.86 ± 2.02 1.86 ± 0.20 4.03 ± 0.47 1.26 ± 0.12 0.72 ± 0.08 0.02 ± 0.01 158.40 ± 16.69
0.40 ± 0.05 1.39 ± 0.12 0.93 ± 0.16 0.09 ± 0.01 0.20 ± 0.03 0.09 ± 0.01 0.10 ± 0.01 0.21 ± 0.03 3.40 ± 0.49
0.03 ± 0.00 0.20 ± 0.01 0.20 ± 0.01 0.12 ± 0.01 0.08 ± 0.01 0.08 ± 0.01 0.04 ± 0.00 3.39 ± 0.21 4.14 ± 0.24
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BDE-209 BDE-100
BDE-183 BDE-99
BDE-154 BDE-47
C oncentrations of ∑P B D E s (ng/g)
C ontributions of P B D E congeners (%)
120
BDE-153 BDE-28
100
80
60
40
15000 Br ain Muscle Liver Lung Fat
12000 9000 6000 3000 0 0
3
6
9
12
Sampling time (months)
20
P Fig. 2. Changes in the mean concentrations of PBDE (ng g1 lipid weigh) in various tissues of the ducks (n = 3) within a year in the semi-field experiment.
0
water sediment
fish
mudsnial
rice
Fig. 1. Congener profiles of polybrominated diphenyl ethers (PBDEs) in water (n = 3), sediment (n = 3), fish (n = 10), mudsnails (n = 10) and rice (n = 3) samples from an e-waste recycling area.
of significant difference in body weight among the ducks collected at four sampling times, biodilution may not be explained as a
contributing factor for the fluctuation of PBDE concentrations in duck tissues with time. In addition, previous studies suggested that laying eggs could cause a decrease in body burden of pollutants in birds (Van den Steen et al., 2009; Akearok et al., 2010). In our study, the ducks sampled in the 3rd month cannot lay eggs because
Table 2 The concentrations (ng g1 lipid weight) of polybrominated diphenyl ethers (mean ± SD) in various tissues and body weight (kg) of ducks (n = 3) at different sampling times. Sampling time
Compound
Muscle
Liver
Lung
Brain
Fat
Body weight
Laying status
0 month
BDE-28 BDE-47 BDE-99 BDE-100 BDE-153 BDE-154 BDE-183 BDE-209 P PBDEs
Contents lists available at ScienceDirect
Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Changes of polybrominated diphenyl ether concentrations in ducks with background exposure level and time Peng-Yan Liu a, Xiao-Ran Chen a,b, Ya-Xian Zhao b, Yuan-Yuan Li b, Xiao-Fei Qin b, Zhan-Fen Qin b,⇑ a b
College of Chemistry and Environmental Science, Hebei University, Baoding, Hebei, China State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
h i g h l i g h t s We conducted a semi-field experiment of ducks in an e-waste recycling site.
P PBDEs increased in fat with time, but fluctuated in other tissues with exposure level. High brominated DE levels increased with time but low brominated DE fluctuated in fat. High brominated DEs seem to have longer half-lives than low brominated DEs in fat.
a r t i c l e
i n f o
Article history: Received 11 October 2013 Received in revised form 2 September 2014 Accepted 11 September 2014 Available online 4 October 2014 Handling Editor: H. Fiedler Keywords: Polybrominated diphenyl ethers Accumulation Ducks Time Exposure levels
a b s t r a c t To reveal what degree bioaccumulation of polybrominated diphenyl ethers (PBDEs) depends on exposure time and other factors, we conducted a semi-field experiment for a year (June 2008–June 2009) in a village in an e-waste recycling site in Taizhou, China. Approximately one hundred of juvenile ducks (Anas domestica Linnaeus) were entrusted to a villager. The ducks lived and forged in a PBDE-polluted pond from the late March to the end of November. Fish and mudsnails that were heavily polluted by PBDEs were main food. In cold days (from December to the middle March), the ducks lived in the villager’ house, and mainly fed on paddy, which contained lower concentrations of PBDEs than fish and mudsnails. The P female ducks were sampled for PBDE analysis every three months. We found that the PBDE concentrations in duck liver, muscle, lung and brain fluctuated greatly with the changes of exposure levels that P were determined by the environment and diets, but the PBDE concentrations in fat tissue increased P PBDE successively with time. Congener analysis demonstrated that the successive increase in the concentrations with time in fat tissue was due to the successive increase in BDE-209, -183 and -153 concentrations, with large fluctuations of low brominated congeners. The results show that PBDE concentrations in liver, muscle, lung and brain tissues heavily depends on exposure levels rather than exposure time. In fat tissue, by contrast, PBDE concentrations (mainly high brominated congeners) slightly depends on exposure levels but heavily depend on time relative to other tissues, implying that high brominated congeners seem to have longer half-lives than low brominated congeners in fat tissue. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Polybrominated diphenyl ethers (PBDEs) are a class of widely used flame retardants in various products, including in textiles, polyurethane foams, thermoplastics and electronic products. Due to high levels of production and application, PBDEs have become ubiquitous environmental pollutants in the environment (Rahman et al., 2001; De Wit, 2002). PBDEs are also found in animals and human tissues because of their hydrophobicity and bioaccumula⇑ Corresponding author. Tel.: +86 10 6291 9177; fax: +86 10 6292 3563. E-mail address: [email protected] (Z.-F. Qin). http://dx.doi.org/10.1016/j.chemosphere.2014.09.037 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
tion (McDonald, 2002; Darnerud, 2008). Animal studies have shown that PBDEs have a wide spectrum of toxicological effects, including thyroid disruption, developmental neurotoxicity, reproduction, immunotoxicity, etc. (Damerud, 2003). There are increasing epidemiological evidences that PBDEs might be associated with some parameters on human health (Chen et al., 2011; Chevrier et al., 2011; Bradman et al., 2012; Eskenazi et al., 2013). Due to bioaccumulation and potential toxicity, thus, PBDEs have aroused great concern. Deca-BDE products have still been produced and used widely, although commercial tetra- and octa-BDEs were added to Annex A of the Stockholm Convention PBDEs in 2009 (Alaee et al., 2003; Covaci et al., 2011).
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Numerous studies have demonstrated that the concentrations of some persistent organic pollutants (POPs), such as polychlorinated biphenyl (PCB), DDT, DDE, increase generally with exposure time or age in biotic tissues (Ronald et al., 1984; Johnson-Restrepo et al., 2005; Munoz-De-Toro et al., 2006; Mcgraw and Waller, 2009; Weijs et al., 2009; Goutner et al., 2011). However, a few of studies have demonstrated that PBDE bioaccumulation seems to increase with age in some biotic tissues (Riget et al., 2006; Kim et al., 2012), whereas some biomonitoring data show no association between PBDEs concentrations and time/age in biotic samples, such as fish (Zhang et al., 2010; Vigano et al., 2011), mammals (Rayne et al., 2004; Muir et al., 2006; Weijs et al., 2009), and humans (Covaci et al., 2008; Harrad and Porter, 2007; Kunisue et al., 2007). Overall, what degree PBDE bioaccumulation depends on exposure time and other factors has been unclear. Taizhou in Zhejiang Province is one of the largest electric waste (e-waste) recycling areas in China. Recycling of e-waste using primitive methods has resulted in heavy PBDE pollution in the environment, although the primitive e-waste recycling has been controlled. In our previous studies, high concentrations of PBDEs were found in soils, sediments, animal tissues and human samples from some e-waste recycling sites in Taizhou (Liang et al., 2008; Yang et al., 2008; Zhao et al., 2009; Liu et al., 2011). To study what degree PBDE bioaccumulation depends on exposure time and other factors, in this study, we conducted a semi-field experiment in an e-waste recycling site in Taizhou to investigate changes in PBDE concentrations in ducks within a year and discuss crucial factors for PBDE accumulation. 2. Materials and methods 2.1. Chemicals BDE-71 and 13C-labled BDE-209 as surrogate standards, and a standard solution of PBDE congeners (EO-5278, BDE-28, -47, -99, -100, -153, -154, -183, -209.) were obtained from Cambridge Isotope Laboratories (USA). Methylene dichloride, n-Hexane, and nonane were of pesticide grade and purchased from Tedia (USA). 2.2. Semi-field experiment on ducks During June 2008–June 2009, we conducted a semi-field experiment in a village in Taizhou, where a mass of e-wastes from abroad as well as home were disassembled and shattered into powder to obtain the usable materials, and discarded e-waste powder was stacked around this village. We purchased about one hundred of one-month-old ducks (Anas domestica Linnaeus) from a farm (no factory and e-waste recycling site in the surrounding) in Zhejiang, and entrusted a villager in this village with them. The villager’ house was approximately 500 m away from the foot of a hill, where a mass of e-waste powder was stacked. There was a pond in the front of the villager’ house. When raining, rain water from the foot of the hill flew into the pond. From the late March to the end of November, the ducks forged in the pond at daytime, and went back to their nest in the villager’ house at night. In the pond, there were a lot of mudsnails and fish, which were good food for the ducks. In cold days, from December to the early March, these ducks lived in the villager’ house, and they mainly fed on paddy. The semi-field experiment began on June 15, when we sampled 3 ducks to examine background PBDE levels in the ducks. On September 15 (the 3rd month), December 15 (the 6th month), March 15 (the 9th month) and the next June 15 (the 12th month), the ducks were sampled for PBDE analysis. Because most ducks died in the first month after the beginning of the experiment, we only sampled 3 female ducks each time. The ducks sampled in the 3rd month never laid eggs, whereas the ducks sampled in the
6th, 9th and 12th month laid eggs. As live ducks were transferred to the laboratory, they were anesthetized and weighed. Liver, lung, muscle, fat and brain tissues were collected. All samples were cleaned with deionized water, weighed and wrapped in aluminum foil twice, and sealed in plastic bags to minimize the possibility of contamination. Then, samples were stored at 20 °C in darkness until analysis. At the beginning of the semi-field experiment, we collected the water, sediment, mudsnail and fish samples from this pond. The water samples (40 L) were filtered through glass fiber filters (0.8 lm pore size) and the filtrate was passed through a XAD-2 resins column (Supelco, Bellfonte, USA), which was pre-extracted with methanol, dichloromethane and hexane, to enrich PBDEs on-site. Then, the XAD-2 resins column and sediment samples were stored in a cool box and transported to laboratory. The eluent was concentrated to about 2 mL in a rotary evaporator. Then the sample solution was transferred to a vial and reduced to a volume of 20 lL under a gentle stream of N2. The sediment samples were stored at 4 °C in darkness until analysis. Sediment samples were taken from approximately the top 2 cm of the sediment and each was constituted by five subsamples. The sediment samples (n = 3) were weighed and wrapped in aluminum foil and sealed in plastic bags on-site. All fish and mudsnails sampled in the pond were transferred to the laboratory for PBDE analysis. In the laboratory, the fish was rinsed with deionized water for three times, weighed and stored at –20 °C in darkness until analysis. After washed with deionized water, live mudsnails were placed in cool fresh water to expel the sediment and sand inside their body. The fresh water was replaced five times within 24 h. After the shells of mudsnails were removed, the samples were washed again, weighed and stored at 20 °C in the dark until analysis. Meanwhile, the rice as duck diet was also collected for PBDE analysis. 2.3. Extraction The tissue samples were freeze-dried and homogenized with anhydrous sodium sulfate, then spiked with BDE-71, and 13 C-labled BDE-209. The samples were subsequently extracted by ultrasonic extraction for twice with 30 mL of n-hexane/dichloromethane (1:1, vol/vol). Then the combined extracts were evaporated to dryness for gravimetric determination of extracted lipid content. The concentrated extract was cleaned by passing through a 15 mm i.d. column, which was packed, from the bottom to top, with 1 g activated silica gel, 3 g alkaline silica gel (33% sodium hydroxide, w/w), 1 g activated silica gel, 4 g acid silica gel (44% concentrated sulfuric acid, w/w), 4 g acid silica gel (22% concentrated sulfuric acid, w/w), 1 g activated silica gel, and 2 cm anhydrous sodium sulfate. The extract was eluted with 100 mL of hexane, concentrated to 2 mL using a rotary evaporator, transferred, and finally concentrated to approximately 0.1 mL under a gentle nitrogen stream. The final extract was transferred to GC vials. Throughout the extraction, cleanup and analysis procedures, the analyte was protected from light by wrapping the containers with aluminum foil or by using amber glassware. 2.4. PBDE analysis All sample extracts were analyzed by Agilent 6890 series gas chromatograph coupled with Agilent 5973 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) using negative chemical ionization (NCI) source in the selected ion monitoring (SIM) mode. The gas chromatography column was DB-5 MS fused silica capillaries (15 m, 250-lm inner diameter, 0.25 lm film thickness). The injector and interface temperature were 265 and 300 °C, respectively. The samples were injected in the pulsed splitless mode.
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Methane was used as the chemical ionization moderating gas and helium as the carrier gas at a flow rate of 1.0 mL min1. The GC oven temperature program was carried out as follows: start at 100 °C, held for 1 min, increased to 200 °C at 10 °C min1, and then to 300 °C at 20 °C min1, held for 20 min. The ions of m/z 79 and 81 were monitored for BDE-28, -47, -100, -99, -154, -153, -183 and m/z 486.7 and 488.7 for BDE-209. 2.5. Quality assurance/quality control To avoid potential sample contamination, cross contamination, and PBDE degradation, proper handling was adopted from sample collection to chemical analysis. One procedural blank was run for every batch of nine samples to check the potential contamination from analysis process. Instrumental quality control was done by regular injection of solvent blanks and standard solutions. Recoveries of surrogate standards BDE-71 and 13C-labled BDE-209 were on average 84 ± 12% and 65 ± 8%, respectively. Analyte value of BDE-209 was corrected for the recovery of 13C-labled BDE-209, and the other congeners were corrected for the recovery of BDE-71. The limit of detection (LOD) was defined as the concentration of analyte in the sample producing a peak with the ratio of signal to noise of 3 (peak-to-peak). Non-detect or below LOD values were estimated as zero for the purpose of calculating totals and means. For the tissue samples, LODs ranged from 0.005 to 0.025 ng g1 wet weight (wet wt) for tri- to hepta- BDE, and 0.03 ng g1 wet weight for deca-BDE.
The average concentrations of PBDEs in fish and mudsnails in the study pond were 158.40 ng g1 wet wt and 3.40 ng g1 wet wt, respectively, while the average PBDE concentration in paddy was 4.14 ng g1 dry weight. The ducks in the semi-wild experiment would absorb much PBDEs via the uptake of fish and mudsnails and direct contact with the water and sediment in the study pond relative to via the uptake of paddy, although we could not measure daily intake of PBDEs by the ducks. There was a large difference in congener profiles of PBDEs between environmental samples and biotic samples from the study pond (Fig. 1). BDE-209 was the dominant congener in sediment and water, in particular, BDE-209 contributed 73.31% of the total PBDEs in water. However, low brominated congeners (BDE-28, -47, -99) were main congeners, whereas high brominated congeners (BDE-209 and BDE-183) were less in fish and mudsnails. The observation is consistent with previous reports that aquatic organisms accumulated much low brominated PBDE congeners and less high brominated congeners from the environment with more high brominated congeners and less low brominated congeners was consistent with previous reports (Sellstrom et al., 1998; Boon et al., 2002; Guo et al., 2008). In paddy samples as duck diets, BDE-209 accounted for approximately 80% of the total PBDEs. Thus, the ducks can absorb much high brominated PBDE congeners by direct contact with the sediment and water in the pond. On the contrary, the ducks can absorb much low brominated congeners by the intake of fish and mudsnails. In addition, the ducks can also absorb much BDE-209 by local paddy. 3.2. Growth of ducks and changes in tissues within a year
2.6. Data analysis All the statistical analysis was performed using SPSS software version 16.0 (SPSS Inc., Chicago, USA). The difference in body weight was tested by one-way analysis of variance. The concentraP tions of PBDEs and each congener in each tissue for adjacent sampling times were analyzed by t-test. P < 0.05 was regarded as statistically significant. 3. Results and discussion 3.1. PBDE levels and congener profiles in the surroundings and diets for ducks Table 1 shows the concentrations of PBDEs in water (dissolved phase and particulate phase), sediment, fish and mudsnails in the study pond as well as paddy samples as duck diets from the ewaste recycling site. The PBDE concentrations in sediment (8551.04 ng g1 dry weight) and water (42.00 ng L1) were much higher than the general levels reported in previous studies (Hale et al., 2003; Law et al., 2003; Yogui and Sericano, 2009). High values of PBDEs detected in water and sediment indicated that e-waste recycling resulted in PBDE pollution in this study area.
P PBDE concentrations in various
The ducks rapidly grew up before the first sampling. No significant difference in body weight was found among ducks collected for four sampling times, despite body weight of the ducks collected for the third time was slightly low (Table 2). The ducks sampled in the 3rd month never laid eggs, but there were some developing eggs in the abdomens. The ducks sampled in the 6th, 9th and 12th month P laid eggs. Fig. 2 and Table 2 shows the PBDE concentrations (ng g1 lipid weight) in fat, liver, lung, muscle, and brain tissues of the ducks. Statistical analysis (t-test) of the data for adjacent P sampling times revealed a rough fluctuation of PBDE concentrations with time. Before the beginning of the semi-field experiment, the ducks had low body burden of PBDEs. After 3 months, the P PBDE concentrations in ducks increased dramatically. The P PBDE concentrations in liver, muscle, lung and brain tissues flucP tuated within a year. In detail, the PBDE concentrations increased in the 6th month relative to the 3rd month, decreased in the 9th month relative to the 6th month, then, increased again during the last 3 months of the semi-field experiment. It is generally believed that the growth possibly results in decreases in pollutant concentrations in the body, so called biodilution (Hammer et al., 1993; Blais et al., 2003). Considering the lack
Table 1 PBDE concentrations (mean ± SD) in water (ng L1, n = 3), sediment (ng g1 dry weight, n = 3), fish (ng g1 wet weight, n = 10), mudsnail (ng g1 wet weight, n = 10) and paddy (ng g1 dry weight, n = 3) samples from an e-waste recycling site. Compound
Water
Sediment
Fish
Mudsnail
Paddy
BDE-28 BDE-4 BDE-99 BDE-100 BDE-153 BDE-154 BDE-183 BDE-209 P PBDEs
0.92 ± 0.08 4.89 ± 0.34 3.80 ± 0.27 0.16 ± 0.01 0.81 ± 0.04 0.28 ± 0.01 0.35 ± 0.02 30.79 ± 2.26 42.00 ± 3.42
263.90 ± 34.21 1972.30 ± 212.37 1805.90 ± 193.38 132.80 ± 15.26 562.00 ± 67.34 189.90 ± 19.47 621.20 ± 78.17 3003.00 ± 483.96 8551.04 ± 921.14
30.83 ± 2.46 100.82 ± 13.48 18.86 ± 2.02 1.86 ± 0.20 4.03 ± 0.47 1.26 ± 0.12 0.72 ± 0.08 0.02 ± 0.01 158.40 ± 16.69
0.40 ± 0.05 1.39 ± 0.12 0.93 ± 0.16 0.09 ± 0.01 0.20 ± 0.03 0.09 ± 0.01 0.10 ± 0.01 0.21 ± 0.03 3.40 ± 0.49
0.03 ± 0.00 0.20 ± 0.01 0.20 ± 0.01 0.12 ± 0.01 0.08 ± 0.01 0.08 ± 0.01 0.04 ± 0.00 3.39 ± 0.21 4.14 ± 0.24
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BDE-209 BDE-100
BDE-183 BDE-99
BDE-154 BDE-47
C oncentrations of ∑P B D E s (ng/g)
C ontributions of P B D E congeners (%)
120
BDE-153 BDE-28
100
80
60
40
15000 Br ain Muscle Liver Lung Fat
12000 9000 6000 3000 0 0
3
6
9
12
Sampling time (months)
20
P Fig. 2. Changes in the mean concentrations of PBDE (ng g1 lipid weigh) in various tissues of the ducks (n = 3) within a year in the semi-field experiment.
0
water sediment
fish
mudsnial
rice
Fig. 1. Congener profiles of polybrominated diphenyl ethers (PBDEs) in water (n = 3), sediment (n = 3), fish (n = 10), mudsnails (n = 10) and rice (n = 3) samples from an e-waste recycling area.
of significant difference in body weight among the ducks collected at four sampling times, biodilution may not be explained as a
contributing factor for the fluctuation of PBDE concentrations in duck tissues with time. In addition, previous studies suggested that laying eggs could cause a decrease in body burden of pollutants in birds (Van den Steen et al., 2009; Akearok et al., 2010). In our study, the ducks sampled in the 3rd month cannot lay eggs because
Table 2 The concentrations (ng g1 lipid weight) of polybrominated diphenyl ethers (mean ± SD) in various tissues and body weight (kg) of ducks (n = 3) at different sampling times. Sampling time
Compound
Muscle
Liver
Lung
Brain
Fat
Body weight
Laying status
0 month
BDE-28 BDE-47 BDE-99 BDE-100 BDE-153 BDE-154 BDE-183 BDE-209 P PBDEs
